This invention relates to a process for determining superoxide dismutase (hereinafter abbreviated to SOD) contained in body fluids, for example, serum and the like.
In the natural world, aerobic living things such as microorganisms, plants and higher animals efficiently obtain energy indispensable for maintenance of their life by utilizing molecular oxygen effectively. It is known that simultaneously with the utilization, various active oxygens are produced and do a variety of injuries to the living bodies. For example, superoxide anion (hereinafter referred to as O.sub.2), which is one of the active oxygens, is extensively harmful to living bodies: for example, it causes inflammation, carcinogenesis, aging, denaturation of nucleic acids, enzymes, lipids and the like.
SOD exists universally in all aerobic living things, is called also superoxide-dismutation enzyme, is an enzyme catalyzing dismutation reaction of O.sub.2 in the living bodies, i.e. 2O.sub.2 +2H.sup.+ .fwdarw.O.sub.2 +H.sub.2 O.sub.2, and protects the living bodies against the toxicity of oxygen.
There are various reports on assay of SOD activity in erythrocyte, leukocyte and tissue slice and on its clinical significance. No sufficient investigation on SOD in serum has been conducted yet because various components such as protein components affect SOD measured values. All the SOD activity determination processes now employed comprise a system for producing O.sub.2 and a system for detecting O.sub.2 and utilize a decrease of the produced amount of O.sub.2 due to acceleration of dismutation of O.sub.2 by SOD.
As the system for producing O.sub.2 in the SOD activity determination processes, there are also, for example, a system in which O.sub.2 is produced by using a superoxidized compound, e.g., potassium superoxide, and a system in which O.sub.2 is produced by reaction of a reduced form coenzyme and an electron carrier. Now there is generally employed a process which mainly comprises acting xanthine oxidase on xanthine. This reaction is shown below: ##STR1##
As the system for detecting O.sub.2, there is, for example, the nitro blue tetrazolium (hereinafter abbreviated to NO.sub.2 -TB) method and the cytochrome c method which utilize the reducing action of O.sub.2, and the epinephrine method, pyrogallol method and 6-hydroxydopamine method which utilize the oxidizing action of O.sub.2.
The principle of these SOD activity determination processes is shown in the following formulae by taking the case of a process using xanthine and xanthine oxidase in the O.sub.2 production system and the NO.sub.2 -TB method in the O.sub.2 detection system: ##STR2##
That is to say, in the O.sub.2 production system, xanthine oxidase, for example, catalyzes the reaction of xanthine with molecular oxygen (O.sub.2) to give O.sub.2, and when SOD is present in the system, dismutation of O.sub.2 is accelerated and O.sub.2 produced becomes O.sub.2 and H.sub.2 O.sub.2. O.sub.2 produced here reduces cytochrome c, NO.sub.2 -TB or the like to subject the same to coloration and oxidizes epinephrine, pyrogallol, 6-hydroxydopamine or the like to subject the same to coloration. Therefore, by utilizing this property, a decrease in absorbance of sample with respect to reagent blank value is measured and SOD activity value is determined. Accordingly, all of these processes are disadvantageous, for example, in that since the reagent blank value is high, the precision of determination is insufficient and the range of determination is narrow. Therefore, there has been desired the advent of an SOD activity determination process which is improved in these points, has higher precision, and has a wider determination range.